Long-term survival for patients with acute myeloid leukemia (AML) remains poor despite advancement in the understanding of AML pathogenesis. However, the addition of differentiation therapy to chemotherapy regimens for patients with acute promyelocytic leukemia (APL) has greatly increased their survival. Experimental evidence suggests that many myeloid leukemias maintain the molecular machinery for cellular maturation. Unfortunately, there are few known pharmacological triggers of differentiation, and these have shown little efficacy in the treatment of other myeloid malignancies. We hypothesize that alternative differentiating agents exist that may have both therapeutic potential and provide insight into the molecular mechanisms of differentiation. Current methods of performing small molecule library screens are limited. To identify myeloid differentiating agents, we designed a new method of high throughput screening (HTS). In this method, a gene expression pattern served as a surrogate for the differentiated phenotype. A gene expression signature distinguishing primary human AML blasts from normal peripheral blood neutrophils or monocytes was determined using DNA microarrays. These signatures were confirmed to be discriminatory in an HL60 cell line model of hematopoietic differentiation. They were then simplified to a 5 gene multiplexed RT-PCR assay. PCR amplicon was detected with a novel method utilizing mass spectrometry. In this proposed project, we will utilize this screening method both to identify new candidate AML differentiating agents and to optimize exposure conditions for dose response and kinetics. The biological activity of these compounds will be evaluated with multiple assays for proliferation and differentiation, and expression profiling will be used to characterize their molecular consequences. Their activity will then be tested in other myeloid cell lines and in primary patient cells. Using those agents with a confirmed differentiation capacity, we will take a multifaceted approach to exploring potential mechanisms of myeloid blast differentiation with DNA microarray technology and mechanism-activity relationship testing. These candidate compounds, as well as an improved understanding of myeloid leukemia differentiation, should help direct us to potential therapeutic agents for AML.